The long-term objective of this renewal application is to continue our investigations into the physiology and cell/molecular biology of the intestinal absorption process of the water-soluble vitamin biotin. We also aim at delineating the cellular and molecular parameters of the intestinal biotin absorption process that are affected by chronic alcohol consumption. Biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Humans and other mammals cannot synthesize biotin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal biotin uptake process. This include characterization of the 5'-regulatory region of the human SLC5A6 gene (the gene that encodes the biotin transporter hSMVT) both in vitro and in vivo, delineation of the mechanisms involved in intracellular trafficking and membrane targeting of hSMVT, and determination of the molecular mechanism involved in the up-regulation of intestinal biotin uptake process in biotin deficiency. Previous in vitro studies from our laboratory using gene-specific siRNA have suggested an important role for hSMVT is biotin uptake by cultured human intestinal epithelial Caco-2 cells. Whether SMVT plays the same role in native intestine in vivo is not clear. Thus, in new preliminary studies aimed at generating conditional SMVT deficient (knockout) mice, we have already generated heterozygote Slc5a6 loxP mice and are currently using them to generating homozygous Slc5a6 loxP mice that will then use to generate global and intestinal-specific SMVT knockout mice. In other preliminary studies, a yeast two-hybrid system was used to screen a human intestinal cDNA library and two proteins (PDZD11 and EPAS1) that interact with hSMVT were identified. Furthermore, molecular evidence was obtained to suggest the involvement of specific histidine residues in hSMVT function. Finally, chronic alcohol feeding of mice was found to lead to a significant reduction in intestinal SMVT mRNA level. Our aims in this proposal are: 1) to characterize the intestinal biotin absorption process in Slc5a6 knockout mice, i.e., to determine the role of SMVT in intestinal biotin absorption in native intestine in vivo;2) To identify accessory protein(s) that interacts with hSMVT and influences its physiology/ biology;3) To investigate structure - function/regulatory aspects of the hSMVT system;and 4) to examine the effect of chronic alcohol consumption on physiological/molecular parameters of intestinal biotin transport. Results of these studies should continue to provide novel and valuable information regarding the physiology/biology of intestinal biotin uptake and the factors that affect the process. This should ultimately assist us in the designing of effective strategies to optimize biotin body homeostasis, especially in conditions associated with biotin deficiency and sub-optimal levels. PUBLIC HEALTH RELEVANCE: Humans and other mammals cannot synthesize the vitamin biotin (an essential micronutrient for normal health) and thus, must obtain it from exogenous sources via intestinal absorption. The aims of this proposal since its inception ten years ago continue to be the delineation of the mechanisms involved in intestinal biotin absorption, how the process is regulated, and what factors affect its function leading to deficiency and sub-optimal levels. Results of these investigations should help in the designing of effective strategies to optimize biotin body levels, especially in conditions associated with biotin deficiency and sub-optimal levels. 1